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Grease matrix as a versatile carrier of proteins for serial crystallography

Nature Methods volume 12, pages 61–63 (2015)Cite this article

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Abstract

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid–binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

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Figure 1: Grease-matrix carrier of protein microcrystals and its extrusion.
Figure 2: Electron density map of lysozyme obtained using microcrystals embedded in grease.

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Acknowledgements

The XFEL experiments were carried out at the BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos. 2012B8036, 2013A8039, 2013A8040, 2013B8044, 2013B8045 and 2014A8032). This work was supported by RIKEN, by the X-ray Free-Electron Laser Priority Strategy Program (MEXT) and partly by Research Acceleration Program of Japan Science and Technology Agency. The sample preparation of FABP3 was supported by the JST-ERATO Murata Lipid Active Structure Project. The authors thank the SACLA beamline staff for technical assistance, K. Diederichs for help with data analysis and A. Nisbet for careful reading of the manuscript.

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Authors and Affiliations

  1. RIKEN SPring-8 Center, Sayo, Japan

    Michihiro Sugahara, Eriko Nango, Mamoru Suzuki, Tomoyuki Tanaka, Tetsuya Masuda, Rie Tanaka, Changyong Song, Jaehyun Park, Takaki Hatsui, Makina Yabashi & So Iwata

  2. Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Japan

    Eiichi Mizohata & Tsuyoshi Inoue

  3. Laboratory of Supramolecular Crystallography, Research Center for Structural and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, Japan

    Mamoru Suzuki

  4. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan

    Tetsuya Masuda

  5. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Sakyo, Japan

    Tatsuro Shimamura, Yoshiki Tanaka, Chiyo Suno, Kentaro Ihara & So Iwata

  6. Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Japan

    Dongqing Pan

  7. Japan Science and Technology Agency (JST)–Exploratory Research for Advanced Technology (ERATO), Lipid Active Structure Project, Toyonaka, Japan

    Keisuke Kakinouchi, Shigeru Sugiyama & Michio Murata

  8. Japan Synchrotron Radiation Research Institute, Sayo, Japan

    Kensuke Tono, Takashi Kameshima & Yasumasa Joti

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  1. Michihiro Sugahara
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Contributions

M. Sugahara introduced grease-matrix extrusion scheme. M. Sugahara, E.M., E.N., M. Suzuki, T.T. and T.M. performed data collection, data processing, structure refinements (lysozyme: M. Sugahara and E.N.; glucose isomerase: E.N. and T.T.; thaumatin: T.M.; FABP3: E.M. and M. Suzuki). E.M., E.N., T.T., T.M., T.S., Y.T., C. Suno, K.I., D.P., K.K., S.S., M.M. and T.I. developed the microcrystal sample preparations and prepared samples (lysozyme: T.S., Y.T., C. Suno, K.I., D.P., E.N. and T.T.; glucose isomerase: E.N. and T.T.; thaumatin: T.M.; FABP3: E.M., K.K., S.S., M.M. and T.I.). E.N. and R.T. designed and developed the injection method. K.T. and M.Y. developed the DAPHNIS. K.T., C. Song, J.P., T.K., T.H., Y.J. and M.Y. developed the SFX systems including injectors. M. Sugahara, E.N. and C. Song wrote the manuscript with input from all the coauthors. S.I. coordinated the project.

Corresponding author

Correspondence to Michihiro Sugahara.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Protein microcrystals used for SFX measurements.

(a) lysozyme, (b) glucose isomerase, (c) thaumatin and (d) FABP3 crystals. Scale bars represent 20 μm.

Supplementary Figure 2 Room-temperature structure of glucose isomerase.

(a) A typical diffraction pattern from an individual microcrystal. Resolution at the edges corresponds to ~1.6 Å. (b) A close-up view of glucose isomerase structure with (2Fo – Fc) electron-density map (contoured at 1.0σ). This figure was drawn with the program PyMol (http://www.pymol.org).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 and Supplementary Tables 1 and 2

Grease matrix carrier of proteins and micro-extrusion.

The crystal solution was dispensed into mineral oil–based grease, and then mixed. The crystal-containing grease was inserted into a dispenser tip. After the tip was centrifuged, the sample was loaded into a syringe. The grease produced a stable flow during the SFX experiment. (AVI 10763 kb)

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Sugahara, M., Mizohata, E., Nango, E. et al. Grease matrix as a versatile carrier of proteins for serial crystallography. Nat Methods 12, 61–63 (2015). https://doi.org/10.1038/nmeth.3172

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